Inhalation of ultrafine particles alters blood leukocyte expression of adhesion molecules in humans.

Ultrafine particles (UFPs; aerodynamic diameter < 100 nm) may contribute to the respiratory and cardiovascular morbidity and mortality associated with particulate air pollution. We tested the hypothesis that inhalation of carbon UFPs has vascular effects in healthy and asthmatic subjects, detectable as alterations in blood leukocyte expression of adhesion molecules. Healthy subjects inhaled filtered air and freshly generated elemental carbon particles (count median diameter 25 nm, geometric standard deviation ~ 1.6), for 2 hr, in three separate protocols: 10 [micro]g/[m.sup.3] at rest, 10 and 25 [micro]g/[m.sup.3] with exercise, and 50 [micro]g/[m.sup.3] with exercise. In a fourth protocol, subjects with asthma inhaled air and 10 [micro]g/[m.sup.3] UFPs with exercise. Peripheral venous blood was obtained before and at intervals after exposure, and leukocyte expression of surface markers was quantitated using multiparameter flow cytometry. In healthy subjects, particle exposure with exercise reduced expression of adhesion molecules CD54 and CD18 on monocytes and CD18 and CD49d on granulocytes. There were also concentration-related reductions in blood monocytes, basophils, and eosinophils and increased lymphocyte expression of the activation marker CD25. In subjects with asthma, exposure with exercise to 10 [micro]g/[m.sup.3] UFPs reduced expression of CD 11 b on monocytes and eosinophils and CD54 on granulocytes. Particle exposure also reduced the percentage of CD[4.sup.+] T cells, basophils, and eosinophils. Inhalation of elemental carbon UFPs alters peripheral blood leukocyte distribution and expression of adhesion molecules, in a pattern consistent with increased retention of leukocytes in the pulmonary vascular bed. Key words: blood leukocytes, human, monocytes, ultrafine particles. Environ Health Perspect 114:51-58 (2006). doi:10.1289/ehp.7962 available via http://dx.doi.org/[Online 20 September 2005]

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Exposure to particulate matter (PM) air pollution is associated with increased respiratory and cardiovascular morbidity and mortality (Peters et al. 2000, 2001a; Pope et al. 2004). Plausible mechanisms explaining the cardiovascular effects of particle exposure have not been clearly defined (Utell et al. 2002). However, recent studies provide evidence that PM exposure is associated with systemic inflammation and changes in vascular function that have been implicated in the pathophysiology of cardiovascular disease, providing clues to possible mechanisms. PM exposure has been associated with increased systolic blood pressure (Ibald-Mulli et al. 2001), plasma viscosity (Peters et al. 1997a), C-reactive protein (Peters et al. 2001b), fibrinogen (Pekkanen et al. 2000), and release of leukocytes from the bone marrow (Mukae et al. 2001; Tan et al. 2000). Increases in ambient concentrations of PM were associated with increased blood leukocyte and platelet counts, as well as fibrinogen (Schwartz 2001). Brook et al. (2002) found evidence for systemic vasoconstriction in resting human subjects exposed to concentrated ambient air particles and ozone.

Ultrafine particles (UFPs), defined as particles with a diameter < 100 nm, have been hypothesized as contributors to cardiovascular effects of PM (Seaton et al. 1995) because, compared with fine particles at similar mass concentrations, they have greater pulmonary deposition efficiency (Chalupa et al. 2004; Daigle et al. 2003), induce more pulmonary inflammation (Li et al. 1999; Oberdorster et al. 1995), have enhanced oxidant capacity (Brown et al. 2001; Li et al. 2003), have a higher propensity to penetrate the epithelium and reach interstitial sites (Stearns et al. 1994), and may even enter the systemic circulation in humans (Nemmar et al. 2002; Oberdorster et al. 2002).

Relatively few epidemiologic studies have examined the health effects of UFP exposure because most ambient air monitoring measures particle mass, and there is relatively poor correlation between particle mass (dominated by fine particles) and particle number (dominated by UFPs). However, a recent study in Erfurt, Germany, found associations between ambient UFPs and mortality (Wichmann et al. 2000). In a study of patients with stable coronary artery disease (Pekkanen et al. 2002), investigators performed repeated exercise tests concurrent with monitoring of ambient particle mass and number counts. Significant independent effects were found for both fine particles and UFPs on the degree of ST-segment depression on the electrocardiogram during exercise.

Asthma, a disease characterized by airway inflammation, confers an increased risk for PM health effects (Atkinson et al. 2001; Lipsett et al. 1997; Tolbert et al. 2000). There is evidence for activation of lung leukocytes and pulmonary vascular endothelium in subjects with asthma, particularly during exacerbations (Ohkawara et al. 1995). Activation of T-lymphocytes with production of "type 2" inflammatory cytokines drives the recruitment and retention of eosinophils in the airway, which contribute to the chronic epithelial injury characteristic of this disease (Corrigan and Kay 1990; Wilson et al. 1992). Treatment with inhaled corticosteroids reduces expression of activation markers CD25 and human leukocyte antigen (HLA)-DR in lung lymphocytes and also reduces HLA-DR expression in blood lymphocytes (Wilson et al. 1994). In asthma, blood CD[4.sup.+] T cells express increased mRNA for interleukin (IL)-4, IL-5, and granulocyte macrophage colony stimulating factor, and IL-5 mRNA expression correlates with asthma severity and eosinophilia (Corrigan et al. 1995). Allergen challenge in subjects with asthma causes a reduction in blood Cd[4.sup.+] T cells (Walker et al. 1992) and an increase in airway Cd[4.sup.+] cells (Virchow et al. 1995). UFP exposure may worsen asthma by further shifting lymphocyte responses to the type 2 phenotype, by further activating resident lymphocytes, by increasing the likelihood that lymphocytes will encounter antigen, and/or by increasing penetration of allergen through an injured epithelium.

We have initiated controlled exposure studies with carbon UFPs in humans, as a surrogate for environmental UFPs, demonstrating that UFPs have a high pulmonary deposition efficiency in healthy subjects (Daigle et al. 2003), which is further increased in subjects with asthma (Chalupa et al. 2004). Exposure to 50 [micro]g/[m.sup.3] carbon UFPs caused a reduction in the pulmonary diffusing capacity for carbon monoxide (Pietropaoli et al. 200%) associated with reductions in the systemic vascular response to increased flow (Pietropaoli et al. 2004a), without significant effects on symptoms, airway inflammation, lung function, or markers of blood coagulation (Pietropaoli et al. 2004c). We hypothesized that inhalation of UFPs alters vascular function, detectable as alterations in blood leukocyte distribution, activation, and expression of adhesion molecules. We further hypothesized that people with asthma, who have airway and systemic inflammation at baseline as well as enhanced UFP deposition, have enhanced susceptibility to these vascular effects. In this article we present detailed analyses of venous blood leukocytes from subjects participating in four separate studies involving carbon UFP exposure: three protocols with varying exposure concentrations in healthy subjects, and one protocol with asthmatic subjects. Some data in this article have been presented previously in abstract form (Frampton et al. 2004).

Materials and Methods

Subjects. Written, informed consent was obtained from all subjects, and the studies were approved by the Research Subjects Review Board of the University of Rochester. Fifty-six never-smoking subjects 18-40 years of age (40 healthy and 16 with asthma) participated and were paid a stipend. Subjects were not studied within 6 weeks of a respiratory infection. Healthy subjects were required to have normal spirometry, a normal 12-lead electrocardiogram, and no history of chronic respiratory disease.

Inclusion criteria for subjects with asthma have been reported previously (Chalupa et al. 2004). These criteria included a consistent clinical history, and either a significant bronchodilator response or airway hyper-responsiveness to methacholine. The severity was consistent with mild intermittent to moderate persistent asthma (National Institutes of Health 1997). Subjects with forced expiratory volume in 1 sec (FE[V.sub.1]) < 70% of predicted at baseline screening, or with > 20% reduction in FE[V.sub.1] after the screening exercise, were excluded.

Study design. Each study used a crossover design in which each subject was exposed to filtered air and to UFPs, so that each subject served as his or her own control. Within each study, the order of air/UFP exposure was randomized, and the randomization was blocked by order of presentation and sex, so that equal numbers of men and women inhaled air first or UFPs first. Exposures were blinded to both subjects and investigators.

Table 1 provides details of each study protocol. The first, UPREST, involved 12 (six female) subjects exposed at rest to approximately 10 [micro]g/[m.sup.3] UFPs or filtered air for 2 hr. The second study protocol, UPDOSE, involved 12 subjects (six female) with three 2-hr exposures with exercise for each subject: approximately 10 [micro]g/[m.sup.3] UFPs, approximately 25 [micro]g/[m.sup.3] UFPs, and filtered air. Subjects exercised on a bicycle ergometer for 15 min of each half hour at an intensity adjusted to increase the minute ventilation to approximately 20 L/mini/[m.sup.2] body surface area. For safety reasons, the order of exposure was randomized in a restricted fashion, so that each subject received the 10-[micro]g/[m.sup.3] exposure before the 25-[micro]g/[m.sup.3]. The third protocol, UP50, involved 16 healthy subjects (eight female) exposed to approximately 50 [micro]g/[m.sup.3] UFPs and air for 2 hr, with intermittent exercise as in the UPDOSE protocol. The final protocol, UPASTHMA, involved 16 subjects with asthma (eight female) exposed to approximately 10 [micro]g/[m.sup.3] UFPs and air for 2 hr, with intermittent exercise as in the UPDOSE protocol. All exposures were separated by at least 2 weeks.

Exposures to either filtered air or UFPs were administered by mouthpiece (with nose clip) for 2 hr, interrupted by a 10-min break after the first hour. Before and at 0, 3.5, and 21 hr after exposure, blood pressure, heart rate, and oxygen saturation by pulse oximetry were measured, and blood was drawn from an antecubital vein. For UP50 and UPASTHMA, measurements were also obtained 45 hr after exposure.

Exposure system. The rationale and design of the exposure facility have been described in detail elsewhere (Chalupa et al. 2002). Briefly, particles [count median diameter ~ 25 nm, geometric standard deviation ~ 1.6] were generated in an argon atmosphere using an electric spark discharge between two graphite electrodes, and then deionized and diluted with filtered air to the desired concentration. Particle number, mass, and size distribution were monitored on both the inspiratory and expiratory sides of the subject. Electronic integration of a pneumotachograph signal provided tidal volume, respiratory frequency, and minute ventilation measurements. Air for the control exposures, and for dilution of the particles, was passed through charcoal and high-efficiency particle filters and was essentially free of particles (0-10 particles/[cm.sup.3]).

Blood leukocyte immunofluorescence analysis. Fresh heparinized whole blood was stained with three monoclonal antibodies: the marker of interest (Table 2) conjugated to fluorescein isothiocyanate, CD14 conjugated to phycoerythrin, and CD45 conjugated to pericidin chlorophyll protein. This permitted determination of the relative expression of adhesion molecules and other markers separately on polymorphonuclear leukocytes (PMNs), eosinophils, lymphocytes, and monocytes. The appropriate isotype control antibodies were run with each experiment to assist in appropriate gate setting. The adhesion markers shown in Table 2 were measured in each of the study protocols, except for CD18, which was measured in UP50 and UPASTHMA only.

Red blood cells were lysed and cells were analyzed on a FACScan flow cytometer (BD Bioscience, San Jose, CA) equipped with a 15-mW argon ion laser emitting at 488 nm. Ten thousand events were collected from each sample in list mode. Standardized fluorescent microbeads (Quantium 24P and 25P; Bangs Laboratories, Fishers, IN) were run with each experiment to convert mean channel numbers to molecules of equivalent soluble fluoro-chrome (MESF) (Gavras et al. 1994). This provided a correction for minor day-to-day instrument variations in fluorescence detection.

Total and differential blood leukocyte and platelet counts were performed in the clinical laboratories of Strong Memorial Hospital, using an automated analyzer (Celldyne 4000; Abott Laboratories, Santa Clara, CA).

Data handling and statistical methods. Data were entered on a desktop computer using Microsoft Excel and analyzed using SAS (SAS Institute Inc., Cary, NC).

UPREST, UPASTHMA, and UP50 used a standard, two-period crossover design in which each subject received both particles and air. Equal numbers of males and females were included. The order of presentation was randomized separately for each sex, with half of each group of subjects receiving each of the two possible orders. UPDOSE used a three-period crossover design in which each subject received air and both 10-[micro]g/[m.sup.3] and 25-[micro]g/[m.sup.3] concentrations of particles. There were then three possible exposure sequences, depending on where in the sequence the air exposure was placed. Equal numbers of subjects were randomly assigned to each sequence.

Repeated-measures analysis of variance (ANOVA) was used (Wallenstein and Fisher 1977), with order of presentation as a between-subjects factor, with exposure and time as within-subject factors. The analysis included tests for period and carryover effects, although the latter were expected to be minimal because of the nature of the exposures and the length of the washout period. In cases where carryover effects were significant, first-period data were examined separately (Jones and Kenward 1989). Each ANOVA included an examination of residuals as a check on the required assumptions of normally distributed errors with constant variance. If these assumptions were not satisfied, data transformations (e.g., square-root transformation for cell counts) were considered. A p-value of 0.05 was required for statistical significance. Data are shown as mean [+ or -] SE, unless otherwise indicated.

Results

Exposure data and subject characteristics. Table 3 shows the exposure parameters and subject characteristics for each protocol. Most of the subjects with asthma were atopic (15 of 16), and most (11 of 16) were not on inhaled steroids, long-acting bronchodilators, or leukotriene inhibitors. All subjects completed every exposure; men and women did not differ in the achieved minute ventilation, adjusted for body surface area. There were no significant effects of UFP exposure on ventilatory parameters or pulmonary function; these results, and UFP deposition, have been published previously (Daigle et al. 2003).

The UPREST protocol, with exposures at rest to 10 [micro]g/[m.sup.3] UFPs, showed no convincing differences between particle and air exposure for leukocyte expression of adhesion molecules or total and differential leukocyte counts.

There were rare statistically significant comparisons, but the significance levels were modest, and the data did not suggest a consistent biologic response. Overall, exposure to 10 [micro]g/[m.sup.3] UFPs at rest had no significant effects on blood leukocytes.

Findings from the three studies involving exercise are described below.

Blood leukocyte expression of adhesion molecules. In these studies, quantitative surface expression of molecules that mediate leukocyte-endothelial interactions served as an indirect indicator of exposure effects on pulmonary vascular endothelial function. The use of flow cytometry with calibrated fluorescent beads allowed quantitation of small changes in surface marker density. Adhesion molecule expression for monocytes and PMNs in the three protocols involving exercise is shown in Tables 4-6.

UPDOSE. UFP exposure caused a concentration-related reduction in monocyte expression of CD54 [intercellular adhesion molecule-1 (ICAM-1) (exposure effect, p = 0.0012); Figure 1]. Expression increased after exposure to filtered air and decreased with 25 [micro]g/[m.sup.3] UFPs, with differences resolving by 21 hr after exposure. Expression of CD62L showed a significant exposure-sex interaction (p = 0.0006; data not shown), with expression increasing in females but decreasing in males relative to air exposure. However, these findings lacked a clear concentration response.

[FIGURE 1 OMITTED]

UP50. Exposure to 50 [micro]g/[m.sup.3] UFPs also reduced expression of CD54 on monocytes (Figure 2A, B), but to a greater extent in males (exposure-sex interaction, p = 0.025). The percentage of monocytes expressing CD54 was also reduced (p = 0.035; data not shown). UFP exposure persistently blunted the airrelated increase in CD 18 expression on monocytes (p = 0.0002; Figure 2C). Expression of CD18 was also reduced on PMNs (Figure 2D), and ANOVA indicated significantly increased CD11a expression on PMNs (exposure-time interaction, p = 0.037; data not shown).

[FIGURE 2A-4D OMITTED]

UPASTHMA. As expected, we found baseline differences between healthy and asthmatic subjects in leukocyte expression of adhesion molecules; these data are shown in Table 7. For example, monocyte expression of CD1 lb, CD54, and CD62L was higher in subjects with asthma than in healthy subjects.

In subjects with asthma, exposure to 10 [micro]g/[m.sup.3] UFPs reduced expression of CD11b on blood monocytes (p = 0.029; Figure 3A) and also reduced expression on eosinophils (p = 0.015; Figure 3B). Expression of CD62L on PMNs increased in males but not females (exposure-sex interaction, p = 0.011; Figure 3C,D). Expression of CD54 on PMNs decreased, with the greatest difference from control at 45 hr after exposure (exposure-time interaction, p = 0.031; data not shown).

[FIGURE 3A-3D OMITTED]

Lymphocyte subsets and activation. There was evidence for increased activated T cells after UFP exposure in healthy subjects. In UPDOSE, CD25 expression on CD[3.sup.+] T cells increased in females, but not in males, early after exposure to 25 [micro]g/[m.sup.3] UFPs (exposure-sex interaction, p = 0.002; Figure 4A, B). In UP50, exposure to 50 [micro]g/[m.sup.3] increased CD25 expression on T cells 0 hr after exposure (p = 0.001 by paired t-test at 0 hr after exposure; p = 0.085 by ANOVA; Figure 4C). There were no other changes in lymphocyte subsets in the healthy subjects.

[FIGURE 4A-4C OMITTED]

In UPASTHMA, CD[4.sup.+] Tcells decreased immediately after exposure to UFPs, compared with air (exposure-time interaction, p = 0.021; Figure 2D). There were no significant effects on other lymphocyte subsets or CD25 expression. However, the percentage of T-lymphocytes expressing the activation marker CD25 was higher in asthmatic subjects than in healthy subjects before exposure (UPASTHMA, 33.0 [+ or -] 3.3%, vs. UPDOSE, 27.0 [+ or -] 2.5%; p = 0.04).

Overall, the data suggest that UFP exposure induces activation (healthy subjects) or sequestration (subjects with asthma) of T-lymphocytes.

Blood leukocyte and platelet counts. In each of the protocols involving exercise (UPDOSE, UP50, and UPASTHMA), consistent postexposure increases were seen in the total leukocyte count and the percentage of PMNs, with decreases in the percentage of eosinophils and monocytes. In the UPDOSE protocol, ANOVA showed a significant exposure-sex interaction for an effect on the percentage of blood monocytes (p = 0.0015). As shown in Figure 5A, B, in females monocytes decreased after exposure to 25 [micro]g/[m.sup.3] and did not return to baseline at 21 hr after exposure. This observation was confirmed when monocyte numbers were analyzed by flow cytometry, using light scatter and CD14 expression (overall effect of UFPs, p = 0.035; exposure-sex interaction, p = 0.002). A significant decrease in blood basophils in females was also seen with both UFP concentrations (exposure-sex interaction, p = 0.015; data not shown).

[FIGURE 5A-5B OMITTED]

Exposure to 50 [micro]g/[m.sup.3] UFPs caused small reductions in the percentage of eosinophils, with a larger effect in females (Figure 5C,D). There were no significant effects on the percentage of blood monocytes, PMNs, or basophils in this protocol.

[FIGURE 5C-5D OMTTED]

In subjects with asthma exposed to 10 [micro]g/[m.sup.3] UFPs, basophils decreased in both men and women at 0 and 3.5 hr after exposure to UFPs, compared with air exposure (exposure-time interaction, p = 0.02; data not shown). The percentage of blood eosinophils as determined by flow cytometry decreased 0 and 3.5 hr after exposure, with greater reductions after UFP exposure than after air (p = 0.049).

UFP exposure did not change platelet counts in any of the exposure protocols.

These data suggest that exposure to UFPs with exercise causes small changes in blood leukocyte differential counts in both healthy and asthmatic subjects.

Discussion

The objective of these studies was to determine whether inhalation of carbon UFPs has vascular effects in healthy subjects, and in subjects with asthma. We postulated that changes in blood leukocyte phenotype and expression of adhesion molecules would serve as a "window" on vascular inflammatory effects after inhalation challenge. Although the specific findings differed among the protocols, all three protocols with exercise showed UFP-associated reductions in expression of adhesion molecules on leukocytes, mainly ICAM-1 (CD54) and the [[beta].sub.2] integrins CD11b and CD18. There were significant differences between healthy and asthmatic subjects in leukocyte expression of adhesion molecules, when measured before exposure (Table 7). For example, blood monocytes from subjects with asthma showed decreased expression of CD 11 a and increased expression of CD11b, CD49d, and CD54 relative to healthy subjects. This may reflect relative activation or priming of circulating leukocytes as a consequence of airway inflammation. In subjects with asthma, inhalation of UFPs reduced expression of CD11b on monocytes and eosinophils (Figure 3) and reduced CD54 expression on PMNs (Table 6).

In addition, the data suggested subtle reductions relative to air exposure in the percentage of blood monocytes, eosinophils, and basophils. There was evidence for activation of CD[4.sup.+] T-lymphocytes in healthy subjects and transient reductions in CD[4.sup.+] T-cell numbers in asthmatic subjects. Sex interactions were seen for some of these changes. A summary of these findings is shown in Table 8.

The findings provide evidence that inhalation of elemental carbon UFPs, with intermittent exercise, causes phenotypic alterations in blood leukocytes at concentrations as low as approximately 10 [micro]g/[m.sup.3] or approximately 2 x 106 particles/[cm.sup.3]. However, the overall nature and direction of the changes do not suggest increased systemic inflammation. This is consistent with the lack of evidence for airway or systemic inflammation that we have reported previously for these studies (Pietropaoli et al. 2004a, 20046.

The reductions in leukocyte subsets and adhesion molecule expression seen in these studies suggest the possibility of leukocyte sequestration or margination in response to UFP exposure. The relative reductions in monocyte, basophil, and eosinophil percentages may result from slightly prolonged transit time through the pulmonary circulation after exposure to UFPs, possibly as a consequence of pulmonary vasoconstriction. The reductions in expression of the adhesion molecules CD54, CD11b, and CD18 are consistent with this hypothesis. Blood leukocytes normally marginate in the lung, requiring several seconds to transit the pulmonary drculation (Doerschuk 2003). PMNs are larger than pulmonary capillaries and must deform in order to transit. The integrins CD1 la and CD11b are expressed as dimers with CD 18 and mediate blood leukocyte recruitment to areas of inflammation and injury via specific receptors on vascular endothdial cells. Activation of monocytes and PMNs increases expression of CD11b and CD18 and decreases cell deformability through actin polymerization (Anderson et al. 2001), slowing transit time. Exercise increases pulmonary blood flow and decreases leukocyte transit time through the pulmonary circulation, leading to mobilization of the pulmonary leukocyte pool into the systemic vascular pool. Van Eeden et al. (1999) have shown that maximal exercise increases the blood leukocyte count and also increases expression of CD11b on peripheral blood PMNs, suggesting that cells expressing higher levels of CD11b preferentially marginate in the pulmonary circulation and are "flushed out" with exercise. Thus, our data are consistent with, but do not prove, increased retention of leukocytes expressing higher levels of adhesion molecules in the pulmonary vascular bed in response to UFP exposure.

Pulmonary vasoconstriction in response to UFP exposure would be expected to delay leukocyte transit through the lung. We have reported (Pietropaoli et al. 2004b) that, in the UP50 protocol, UFP exposure caused reductions in the diffusing capacity for carbon monoxide, without effects on the forced vital capacity, consistent with reduced vascular perfusion or reduced ventilation/perfusion matching. We also reported preliminary findings (Pietropaoli et al. 2004a) of subtle but significant effects on systemic flow-mediated vascular dilatation, and a decrease in blood nitrate levels, suggesting the vascular effects may result from decreased nitric oxide availability. Batalha et al. (2002) have shown pulmonary vasoconstriction in rats exposed to concentrated ambient fine particles.

Alternative mechanisms for reductions in leukocyte and their surface markers include a) direct effects of UFPs on blood leukocytes, reducing surface marker expression through shedding, redistribution, or internalization; b) indirect effects of mediators released by vascular endothelium, such as nitric oxide, which has anti-inflammatory properties (Lefer 1997), reduces endothelial expression of adhesion molecules via inhibition of nuclear factor [kappa]B activation, and reduces monocyte adhesion to endothelium (De Caterina et al. 1995); c) adsorption of soluble cytokines, such as transforming growth factor-13, onto the surface of the particles, reducing inflammatory effects (Kim et al. 2003); d) recruitment of immature leukocytes from the bone marrow in response to UFP inhalation, as has been suggested in previous studies of fine particle exposure (Tan et al. 2000); and e) selective toxicity of UFPs for activated blood leukocytes, inducing apoptosis of specific cell subsets.

The two protocols with exercise in healthy subjects showed increased expression of CD25 on blood T-lymphocytes, and subjects with asthma showed a transient reduction in CD[4.sup.+] lymphocytes after UFP exposure. CD25 is the 0t-chain of the IL-2 receptor; IL-2 promotes lymphocyte proliferation. We found that lymphocyte CD25 expression was higher in subjects with asthma than in healthy subjects, confirming previous observations that people with asthma have a higher percentage of circulating activated T-lymphocytes (Corrigan and Kay 1990), which may explain why UFP exposure did not increase it further in these subjects. The rapid and transient nature of the reduction in CD[4.sup.+] T cells suggests redistribution or margination of cells, as postulated above for other blood leukocytes.

The changes in response to carbon UFP exposure reported in these studies were generally small and would not be expected to adversely affect healthy and mildly asthmatic subjects similar to those studied. However, ambient UFPs contain reactive organic species and transition metals that may induce greater effects than those we observed. People with severely compromised cardiovascular status may experience adverse effects from even small changes in vascular homeostasis. Furthermore, prolonged, repeated exposures may hasten the progression of atherosclerosis, as has been suggested in an epidemiology study of fine particle exposure (Ktinzli et al. 2005).

The UFP number concentrations used in these studies are higher than UFP background concentrations but are relevant to episodic levels seen in specific situations. UFPs are always present in ambient air, with background urban levels in the range of 40,000-50,000 particles/[cm.sup.3] or estimated mass concentrations of 3-4 [micro]g/[m.sup.3] (Peters et al. 1997b). Episodic increases have been documented to 300,000 particles/[cm.sup.3], or estimated to approximately 50 [micro]g/[m.sup.3] UFPs as an hourly average (Brand et al. 1991, 1992). Particle numbers inside a vehicle on a major highway reached 107 particles/[cm.sup.3] (Kittelson et al. 2001), comparable with the highest concentrations used in our studies.

Although not specifically powered to detect sex differences, these studies were designed to include an analysis of sex interactions with the effects of UFP exposure. In the UPDOSE protocol, females showed greater decreases in blood monocytes (Figure 5A) and basophils and greater increases lymphocyte CD25 expression (Figure 4A) compared with males. Females also showed decreased eosinophils in the UP50 protocol (Figure 5C). In UPASTHMA, expression of L-selectin (CD62L) on PMNs was increased in males (Figure 3B). It is possible that males and females differ in their cardiovascular responses to particle exposure. There are known sex differences in leukocyte function and cardiovascular responses, based in part on hormonal influences. For example, females have a higher percentage of CD[4.sup.+] T cells and a higher CD[4.sup.+]:CD[8.sup.+] ratio than do males. Stimulated blood monocytes from females produce more prosraglandin [E.sub.2] (Leslie and Dubey 1994) and less tumor necrosis factor-[alpha] and IL-6 (Schwarz et al. 2000) than those from males. There are also sex differences in endothelial function and antioxidant defenses that may affect vascular response to inhaled challenge. However, we do not feel that these studies have convincingly established or excluded significant sex differences in responses to carbon UFPs.

There are limitations to this study. First, our particles were laboratory-generated elemental carbon, without significant organic species, metals, oxides, nitrates, or sulfates. The findings of these studies may not be representative of exposure to ambient particles, which are a mix of ultrafine, fine, and coarse particles, with reactive organic species, metals, and oxidants in addition to elemental carbon. These and other chemical species may enhance pulmonary and vascular effects. Second, each protocol involved a fairly large number of measurements, and some statistically significant changes may have been chance related. Our approach was to consider results that showed consistency within and across protocols and to discount findings of isolated statistical significance that were not supported by other data. The observations of UFP effects on leukocyte distribution and surface marker expression meet those criteria.

Conclusions

Overall, the findings from these studies provide evidence that inhalation of carbon UFPs, with exercise, reduces peripheral blood monocytes, eosinophils, and basophils and reduces expression of some adhesion molecules on monocytes and PMNs. When considered in light of other evidence, the leukocyte changes may be a consequence of endothelial activation or vasoconstriction in the pulmonary and/or systemic circulation.

This work was supported by contract 98-19 from the Health Effects Institute (HEI); U.S. Environmental Protection Agency (EPA) assistance agreements R826781-01 and R827354-01; grants RO1 ES011853, RR00044, and ES01247 from the National Institutes of Health; and grant 4913-ERTER-ES-99 from the New York State Energy Research and Development Authority.

Some of the research described in this article was conducted under contract to the HEI, an organization jointly funded by the U.S. EPA (assistance agreement X-812059) and automotive manufacturers. The contents of this article do not necessarily reflect the views of the HEI, nor do they necessarily reflect the policies of the U.S. EPA or of automotive manufacturers.

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The authors declare they have no competing financial interests.

Address correspondence to M.W. Frampton, University of Rochester Medical Center, 601 Elmwood Ave., Box 692, Rochester, NY 14642-8692 USA. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

Mark W. Frampton, (1,2) Judith C. Stewart, (1) Gunter Oberdorster, (2) Paul E. Morrow, (2) David Chalupa, (1) Anthony P. Pietropaoli, (1) Lauren M. Frasier, (1) Donna M. Speers, (1) Christopher Cox, (3) Li-Shan Huang, (4) and Mark J. Utell (1,2)

(1) Department of Medicine, and (2) Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York, USA; (3) Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA; 4) Department of Biostatistics, University of Rochester School of Medicine, Rochester, New York, USA

Received 25 January 2005; accepted 20 September 2005.

Table 1. Study design (mean [+ or -] SD).

                           UPREST               UPDOSE

No. of subjects              12                   12
Subject age          30.1 [+ or -] 8.9    26.9 [+ or -] 5.8
 (years)
FE[V.sub.1]         103.8 [+ or -] 8.0   106.3 [+ or -] 16.6
 (% predicted)
Nominala particle         0, 10                0, 10, 25
 mass ([micro]g/
 [m.sup.3])
Rest/exercise              Rest          Intermittent exercise

                           UP50                UPASTHMA

No. of subjects              16                     16
Subject age          26.9 [+ or -] 6.5      23.0 [+ or -] 2.7
 (years)
FE[V.sub.1]         102.8 [+ or -] 9.5      97.6 [+ or -] 5.0
 (% predicted)
Nominala particle         0, 50                  0, 10
 mass ([micro]g/
 [m.sup.3])
Rest/exercise       Intermittent exercise   Intermittent exercise

(a) The target mass concentration of UFPs for each protocol.

Table 2. Leukocyte markers measured in each protocol.

Cluster
designation   Name            Source (clone)

CD3                           BD Bioscience (a) (SK7)
CD4                           BD Bioscience (SK3)
CD8                           BD Bioscience (SK1)
CD11 (a)      Leukocyte       GenTrak (b) (38) or
              function        Coulter (c) (25.3.1)
              antigen-1
CD11 (b)      Mac-1           Ancell (d) (ICRF44)
CD18 (e)                      Pharmigena (6.7) o
                              BD Bioscience (1-130)
CD25          Tac             BD Bioscience (2A3)
CD49d         Very late       Serotec (f) (44H6)
              antigen-
              [alpha]4
CD54          Intercellular   Southern Biotechnology (g)
              adhesion        (15.2)
              molecule-1
CD62L         L-selectin      Coulter (DREG56) or
                              Pharmipen (DREG56)

Cluster
designation   Description

CD3           Marker of T-lymphocytes
CD4           Marker of T-helper lymphocytes
CD8           Marker of T-cytotoxic lymphocytes
CD11 (a)      Part of [[beta].sub.2] integrin adhesion molecule complex
CD11 (b)      Subunit of complement receptor 3, part of [[beta].sub.2]
              integrin adhesion molecule complex
CD18 (e)      Part of [[beta].sub.2] adhesion molecule complex with
              CD11a and CD11b
CD25          Epitope of IL-2 receptor, activation marker on
              lymphocytes
CD49d         Part of [[beta].sub.2] integrin adhesion molecule complex
CD54          Adhesion molecule
CD62L         Adhesion molecule

(a) San Jose, CA. (b) Plymouth Meeting, PA. (c) Miami, FL. (e) Bayport,
MN. (e) Measured in UP50 and UPASTHMA only. (f) Raleigh, NC.
(g) Birmingham, AL.

Table 3. Exposure parameters  (mean [+ or -] SD).

                            UPREST              UPDOSE

Nominal particle             10                    10
 mass ([micro]g/
 [m.sup.3])
Measured particle   10.00 [+ or -] 2.14   13.87 [+ or -]  4.02
 mass ([micro]g/
 [m.sup.3])
Particle number      1.88 [+ or -] 0.09    2.04 [+ or -]  0.07
 (x [10.sup.6]
 particles/
 [cm.sup.3])
CMD(nm)              27.3 [+ or -] 2.5     25.2 [+ or -] 1.7
GSD                  1.62 [+ or -] 0.02    1.60 [+ or -] 0.02

                            UPDOSE                UP50

Nominal particle             25                    50
 mass ([micro]g/
 [m.sup.3])
Measured particle   28.46 [+ or -] 5.13   49.97 [+ or -] 3.88
 mass ([micro]g/
 [m.sup.3])
Particle number      6.96 [+ or -] 0.10   10.79 [+ or -] 1.66
 (x [10.sup.6]
 particles/
 [cm.sup.3])
CMD(nm)              26.5 [+ or -] 1.5     27.9 [+ or -] 2.2
GSD                  1.60 [+ or -] 0.02    1.65 [+ or -] 0.02

                           UPASTHMA

Nominal particle             10
 mass ([micro]g/
 [m.sup.3])
Measured particle   11.08 [+ or -] 3.11
 mass ([micro]g/
 [m.sup.3])
Particle number      2.20 [+ or -] 0.10
 (x [10.sup.6]
 particles/
 [cm.sup.3])
CMD(nm)              23.1 [+ or -] 1.6
GSD                  1.64 [+ or -] 0.01

Abbreviations: CMD, count median diameter; GSD, geometric standard
deviation.

Table 4. Adhesion molecule expression on monocytes  and PMNs, UPDOSE
protocol (mean [+ or -] SE, MESF).

             Exposure
            ([micro]g/
           [m.sup.3]a)          Baseline                  0 hr

Monocytes
CD11a         Air        64,429 [+ or -] 2,072   62,483 [+ or -] 2,140
              UFP 10     63,818 [+ or -] 4,109   59,900 [+ or -] 2,493
              UFP 25     62,835 [+ or -] 2,644   56,207 [+ or -] 5,436
CD11b         Air        19,034 [+ or -] 986     19,497 [+ or -] 997
              UFP 10     17,632 [+ or -] 990     17,287 [+ or -] 1,171
              UFP 25     19,056 [+ or -] 1,214   17,769 [+ or -] 922
CD49d         Air        14,222 [+ or -] 1,000   13,562 [+ or -] 854
              UFP 10     13,634 [+ or -] 1,029   12,587 [+ or -] 694
              UFP 25     13,590 [+ or -] 839     12,779 [+ or -] 574
CD54          Air        12,188 [+ or -] 319     13,096 [+ or -] 519
              UFP 10     12,541 [+ or -] 469     12,470 [+ or -] 583
              UFP 25     13,717 [+ or -] 686     12,591 [+ or -] 584
CD62L         Air        43,970 [+ or -] 3,212   34,937 [+ or -] 3,519
              UFP 10     38,953 [+ or -] 3,465   30,281 [+ or -] 2,510
              UFP 25     41,357 [+ or -] 4,453   33,134 [+ or -] 2,940

PMNs
CD11a         Air        28,637 [+ or -] 1,073   28,613 [+ or -] 1,228
              UFP 10     29,124 [+ or -] 1,073   26,216 [+ or -] 1,160
              UFP25      28,444 [+ or -] 1,397   27,939 [+ or -] 1,151
CD11b         Air        18,467 [+ or -] 1,117   18,837 [+ or -] 1,223
              UFP 10     16,728 [+ or -] 907     15,997 [+ or -] 1,175
              UFP25      19,778 [+ or -] 2,671   15,671 [+ or -] 1,179
CD49d         Air         7,422 [+ or -] 593      6,572 [+ or -] 542
              UFP 10      7,007 [+ or -] 561      6,172 [+ or -] 559
              UFP 25      6,681 [+ or -] 465      6,031 [+ or -] 442
CD54          Air         4,792 [+ or -] 279      4,500 [+ or -] 280
              UFP 10      4,953 [+ or -] 271      4,292 [+ or -] 242
              UFP 25      4,771 [+ or -] 321      4,084 [+ or -] 216
CD62L         Air        66,179 [+ or -] 3,910   59,419 [+ or -] 4,413
              UFP 10     60,976 [+ or -] 4,340   57,202 [+ or -] 4,515
              UFP 25     66,145 [+ or -] 4,231   60,044 [+ or -] 5,434

             Exposure
            ([micro]g/
           [m.sup.3]a)            3.5 hr                  21 hr

Monocytes
CD11a         Air        62,571 [+ or -] 1,689   65,682 [+ or -] 2,435
              UFP 10     59,190 [+ or -] 3,063   65,249 [+ or -] 2,518
              UFP 25     59,635 [+ or -] 2,404   63,008 [+ or -] 2,126
CD11b         Air        21,076 [+ or -] 1,653   20,901 [+ or -] 1,912
              UFP 10     18,335 [+ or -] 1,501   19,391 [+ or -] 1,185
              UFP 25     22,059 [+ or -] 4,697   22,669 [+ or -] 3,357
CD49d         Air        13,717 [+ or -] 880     13,989 [+ or -] 964
              UFP 10     12,946 [+ or -] 706     13,059 [+ or -] 797
              UFP 25     12,372 [+ or -] 683     13,542 [+ or -] 935
CD54          Air        13,908 [+ or -] 645     13,307 [+ or -] 823
              UFP 10     12,855 [+ or -] 592     13,110 [+ or -] 781
              UFP 25     13,533 [+ or -] 856     14,482 [+ or -] 991
CD62L         Air        37,600 [+ or -] 3,391   37,399 [+ or -] 3,716
              UFP 10     32,409 [+ or -] 1,719   36,356 [+ or -] 3,207
              UFP 25     34,676 [+ or -] 3,234   39,168 [+ or -] 4,196

PMNs
CD11a         Air        28,793 [+ or -] 1,183   28,867 [+ or -] 1,503
              UFP 10     26,260 [+ or -] 985     27,620 [+ or -] 923
              UFP25      27,817 [+ or -] 1,137   27,157 [+ or -] 1,411
CD11b         Air        21,427 [+ or -] 3,186   21,189 [+ or -] 2,383
              UFP 10     16,049 [+ or -] 1,112   21,169 [+ or -] 2,394
              UFP25      20,461 [+ or -] 3,457   18,653 [+ or -] 1,760
CD49d         Air         6,404 [+ or -] 498      6,098 [+ or -] 686
              UFP 10      6,173 [+ or -] 423      6,340 [+ or -] 650
              UFP 25      5,677 [+ or -] 446      5,925 [+ or -] 470
CD54          Air         4,586 [+ or -] 246      4,457 [+ or -] 243
              UFP 10      4,608 [+ or -] 424      4,435 [+ or -] 213
              UFP 25      4,122 [+ or -] 215      4,417 [+ or -] 230
CD62L         Air        64,867 [+ or -] 4,303   59,671 [+ or -] 5,970
              UFP 10     56,621 [+ or -] 4,636   60,626 [+ or -] 4,180
              UFP 25     59,625 [+ or -] 4,296   61,184 [+ or -] 4,054

             Exposure
            ([micro]g/
           [m.sup.3]a)   ANOVA

Monocytes
CD11a         Air
              UFP 10
              UFP 25
CD11b         Air
              UFP 10
              UFP 25
CD49d         Air
              UFP 10
              UFP 25
CD54          Air        Exposure
              UFP 10     p = 0.001
              UFP 25
CD62L         Air        Exposure x sex
              UFP 10     p = 0.006
              UFP 25

PMNs
CD11a         Air
              UFP 10
              UFP25
CD11b         Air
              UFP 10
              UFP25
CD49d         Air        Exposure x sex
              UFP 10     p = 0.007
              UFP 25
CD54          Air
              UFP 10
              UFP 25
CD62L         Air
              UFP 10
              UFP 25

Table 5. Adhesion molecule expression on monocytes and PMNs, UP50
protocol (mean [+ or -] SE, MESF).

            Exposure          Baseline                   0 hr

Monocytes
CD11 (a)       Air     65,882 [+ or -] 3,277   66,463 [+ or -] 2,934
               UFP     69,090 [+ or -] 3,146   68,680 [+ or -] 2,935
CD11 (b)       Air     16,840 [+ or -] 899     20,104 [+ or -] 905
               UFP     18,365 [+ or -] 1,153   19,733 [+ or -] 1,206
CD18           Air     62,675 [+ or -] 2,948   68,897 [+ or -] 2,942
               UFP     67,246 [+ or -] 2,751   67,175 [+ or -] 2,582
CD49 (d)       Air     16,334 [+ or -] 939     16,588 [+ or -] 859
               UFP     16,643 [+ or -] 938     16,445 [+ or -] 874
CD54           Air      9,637 [+ or -] 1,431   10,654 [+ or -] 1,668
               UFP     10,526 [+ or -] 1,715   11,095 [+ or -] 1,782
CD62L          Air     58,551 [+ or -] 3,188   50,197 [+ or -] 3,410
               UFP     57,666 [+ or -] 3,519   49,307 [+ or -] 3,261

PMNs
C011 (a)       Air     30,921 [+ or -] 851     30,934 [+ or -] 862
               UFP     31,569 [+ or -] 1,014   32,158 [+ or -] 1,055
CD11 (b)       Air     16,406 [+ or -] 628     18,053 [+ or -] 934
               UFP     16,678 [+ or -] 830     19,155 [+ or -] 1,953
CD18           Air     34,919 [+ or -] 1,335   36,961 [+ or -] 1,352
               UFP     36,010 [+ or -] 1,032   37,687 [+ or -] 1,810
CD49 (d)       Air      6,455 [+ or -] 412      6,345 [+ or -] 264
               UFP      6,186 [+ or -] 335      6,252 [+ or -] 330
CD54           Air      8,182 [+ or -] 584      8,339 [+ or -] 484
               UFP      8,524 [+ or -] 427      9,071 [+ or -] 545
CD62L          Air     87,437 [+ or -] 4,510   88,596 [+ or -] 3,485
               UFP     92,053 [+ or -] 4,760   89,783 [+ or -] 4,262

            Exposure            3.5 hr                  21 hr

Monocytes
CD11 (a)       Air     65,658 [+ or -] 2,963   69,888 [+ or -] 2,853
               UFP     66,222 [+ or -] 2,696   69,813 [+ or -] 2,835
CD11 (b)       Air     19,938 [+ or -] 835     18,728 [+ or -] 1,092
               UFP     18,531 [+ or -] 952     18,389 [+ or -] 932
CD18           Air     67,872 [+ or -] 2,780   68,661 [+ or -] 2,749
               UFP     66,277 [+ or -] 2,488   67,307 [+ or -] 2,768
CD49 (d)       Air     17,371 [+ or -] 954     16,951 [+ or -] 9,571
               UFP     17,182 [+ or -] 965     17,282 [+ or -] 909
CD54           Air     11,198 [+ or -] 1,728    9,969 [+ or -] 1,639
               UFP     10,889 [+ or -] 1,871   10,352 [+ or -] 1,791
CD62L          Air     48,580 [+ or -] 3,027    9,699 [+ or -] 1,557
               UFP     50,241 [+ or -] 2,848   56,880 [+ or -] 3,515

PMNs
C011 (a)       Air     31,339 [+ or -] 960     31,683 [+ or -] 944
               UFP     31,652 [+ or -] 912     31,751 [+ or -] 927
CD11 (b)       Air     17,262 [+ or -] 678     17,355 [+ or -] 869
               UFP     17,076 [+ or -] 777     18,014 [+ or -] 713
CD18           Air     36,486 [+ or -] 1,286   35,907 [+ or -] 1,226
               UFP     36,255 [+ or -] 1,060   35,316 [+ or -] 983
CD49 (d)       Air      6,399 [+ or -] 279      6,145 [+ or -] 204
               UFP      6,362 [+ or -] 340      6,284 [+ or -] 305
CD54           Air      8,973 [+ or -] 552      8,114 [+ or -] 415
               UFP      8,668 [+ or -] 458      8,501 [+ or -] 402
CD62L          Air     88,617 [+ or -] 4,056   87,244 [+ or -] 3,362
               UFP     90,736 [+ or -] 4,227   89,363 [+ or -] 3,898

            Exposure            45 hr          ANOVA

Monocytes
CD11 (a)       Air     71,292 [+ or -] 2,885
               UFP     71,773 [+ or -] 3,132
CD11 (b)       Air     18,364 [+ or -] 993
               UFP     18,369 [+ or -] 815
CD18           Air     68,963 [+ or -] 3,187   Exposure
               UFP     68,754 [+ or -] 3,052   p = 0.0002
CD49 (d)       Air     17,126 [+ or -] 1,079
               UFP     17,484 [+ or -] 1,167
CD54           Air      9,827 [+ or -] 1,687   Exposure x sex
               UFP     10,339 [+ or -] 1,811   p = 0.025
CD62L          Air     59,189 [+ or -] 2,271
               UFP     58,283 [+ or -] 3,020

PMNs
C011 (a)       Air     31,712 [+ or -] 937     Exposure x time
               UFP     32,130 [+ or -] 921     p = 0.037
CD11 (b)       Air     17,525 [+ or -] 848
               UFP     17,545 [+ or -] 694
CD18           Air     35,868 [+ or -] 1,450   Exposure
               UFP     35,682 [+ or -] 1,087   p = 0.023
CD49 (d)       Air      6,070 [+ or -] 203
               UFP      6,114 [+ or -] 258
CD54           Air      8,072 [+ or -] 383
               UFP      8,446 [+ or -] 389
CD62L          Air     89,489 [+ or -] 2,648
               UFP     94,055 [+ or -] 4,598

Table 6. Adhesion molecule expression on monocytes and PMNs, UPASTHMA
protocol (mean [+ or -] SE, MESF).

            Exposure          Baseline                  0 hr

Monocytes
CD11 (a)      Air      21,179 [+ or -] 4,120   20,442 [+ or -] 3,989
              UFP      32,102 [+ or -] 7,076   30,277 [+ or -] 6,791
CD11 (b)      Air      25,022 [+ or -] 2,822   31,626 [+ or -] 5,969
              UFP      26,958 [+ or -] 4,112   25,452 [+ or -] 4,611
CD18          Air      85,586 [+ or -] 6,773   87,234 [+ or -] 8,882
              UFP      84,999 [+ or -] 7,252   81,131 [+ or -] 7,931
CD49 (d)      Air      17,172 [+ or -] 731     16,739 [+ or -] 925
              UFP      18,378 [+ or -] 865     16,967 [+ or -] 873
CD54          Air      19,102 [+ or -] 1,386   19,432 [+ or -] 1,430
              UFP      20,673 [+ or -] 2,009   20,438 [+ or -] 2,088
CD62L         Air      45,571 [+ or -] 2,571   39,446 [+ or -] 2,652
              UFP      51,939 [+ or -] 5,305   43,483 [+ or -] 4,955

PMNs
CD11 (a)      Air      10,540 [+ or -] 1,775   10,010 [+ or -] 1,771
              UFP      14,562 [+ or -] 2,749   14,161 [+ or -] 2,679
CD11 (b)      Air      24,078 [+ or -] 2,783   26,353 [+ or -] 3,578
              UFP      23,819 [+ or -] 2,343   22,792 [+ or -] 3,224
CD18          Air      48,861 [+ or -] 3,054   47,564 [+ or -] 3,026
              UFP      46,982 [+ or -] 2,925   44,465 [+ or -] 2,676
CD49 (d)      Air       5,342 [+ or -] 211      5,122[+ or -]2 28
              UFP       5,499 [+ or -] 315      4,964 [+ or -] 212
CD54          Air       5,631 [+ or -] 230      5,348 [+ or -] 236
              UFP       6,262 [+ or -] 451      5,759 [+ or -] 453
CD62L         Air      78,859 [+ or -] 3,812   69,825 [+ or -] 3,978
              UFP      79,315 [+ or -] 6,332   75,646 [+ or -] 6,405

            Exposure           3.5 hr                   21 hr

Monocytes
CD11 (a)      Air      19,336 [+ or -] 4,042   21,126 [+ or -] 5,569
              UFP      29,592 [+ or -] 6,630   30,468 [+ or -] 6,809
CD11 (b)      Air      26,553 [+ or -] 3,319   26,345 [+ or -] 3,456
              UFP      25,742 [+ or -] 4,241   24,498 [+ or -] 4,199
CD18          Air      82,899 [+ or -] 6,465   82,697 [+ or -] 7,370
              UFP      81,297 [+ or -] 9,950   82,028 [+ or -] 6,767
CD49 (d)      Air      16,013 [+ or -] 616     16,627 [+ or -] 837
              UFP      17,138 [+ or -] 919     17,715 [+ or -] 877
CD54          Air      18,285 [+ or -] 1,248   19,043 [+ or -] 1,410
              UFP      19,861 [+ or -] 1,934   20,014 [+ or -] 1,853
CD62L         Air      41,214 [+ or -] 2,703   45,100 [+ or -] 2,847
              UFP      42,198 [+ or -] 3,954   46,105 [+ or -] 4,023

PMNs
CD11 (a)      Air      10,107 [+ or -] 1,837   10,986 [+ or -] 2,830
              UFP      13,790 [+ or -] 2,780   13,765 [+ or -] 2,727
CD11 (b)      Air      25,211 [+ or -] 2,533   25,199 [+ or -] 2,072
              UFP      25,376 [+ or -] 2,984   22,085 [+ or -] 2,479
CD18          Air      45,449 [+ or -] 2,457   45,303 [+ or -] 2,719
              UFP      43,512 [+ or -] 3,174   44,599 [+ or -] 2,862
CD49 (d)      Air       5,090 [+ or -] 162      4,805 [+ or -] 248
              UFP       4,887 [+ or -] 210      4,783 [+ or -] 234
CD54          Air       5,234 [+ or -] 222      5,433 [+ or -] 277
              UFP       5,604 [+ or -] 458      5,535 [+ or -] 399
CD62L         Air      71,796 [+ or -] 3,691   72,829 [+ or -] 4,711
              UFP      70,468 [+ or -] 4,961   74,971 [+ or -] 5,500

            Exposure            45 hr          ANOVA

Monocytes
CD11 (a)      Air      21,407 [+ or -] 5,550
              UFP      29,751 [+ or -] 6,640
CD11 (b)      Air      27,703 [+ or -] 3,228   Exposure
              UFP      25,814 [+ or -] 3,502   p = 0.029
CD18          Air      85,455 [+ or -] 7,819
              UFP      77,346 [+ or -] 7,334
CD49 (d)      Air      16,856 [+ or -] 771
              UFP      17,327 [+ or -] 879
CD54          Air      19,281 [+ or -] 1,319
              UFP      19,284 [+ or -] 1,491
CD62L         Air      44,329 [+ or -] 2,870
              UFP      45,608 [+ or -] 4,271

PMNs
CD11 (a)      Air      11,199 [+ or -] 2,953
              UFP      13,710 [+ or -] 2,652
CD11 (b)      Air      30,893 [+ or -] 4,350
              UFP      22,781 [+ or -] 1,886
CD18          Air      50,312 [+ or -] 5,429
              UFP      43,470 [+ or -] 3,006
CD49 (d)      Air       4,923 [+ or -] 185
              UFP       4,950 [+ or -] 241
CD54          Air       5,635 [+ or -] 239     Exposure x time
              UFP       5,660 [+ or -] 398     p = 0.031
CD62L         Air      72,429 [+ or -] 4,184   Exposure x sex
              UFP      74,541 [+ or -] 5,925   p = 0.011

Table 7. Blood leukocyte marker expression at
baseline that differed between asthmatic and
healthy subjects (mean [+ or -] SE, MESF).

                     Healthy (a)             Asthma           p-Value

Lymphocytes
CD11 (a)      41,710 [+ or -] 1,844   14,575 [+ or -] 4,161   < 0.001
CD11 (b)       1,460 [+ or -] 67       1,784 [+ or -] 107       0.017
CD49 (d)       8,168 [+ or -] 335     10,486 [+ or -] 324     < 0.001
CD54           2,381 [+ or -] 69       2,964 [+ or -] 155       0.003
Monocytes
CD11 (a)      64,155 [+ or -] 4,041   26,220 [+ or -] 5,260   < 0.001
CD11 (b)      17,944 [+ or -] 915     25,047 [+ or -] 2,751     0.025
CD49 (d)      13,556 [+ or -] 915     17,089 [+ or -] 642       0.005
CD54          12,314 [+ or -] 401     17,942 [+ or -] 1,065   < 0.001
PMNs
CD11 (a)      28,358 [+ or -] 904     12,753 [+ or -] 2,276   < 0.001
CD11 (b)      16,868 [+ or -] 1,055   24,178 [+ or -] 2,705     0.021
C049 (d)       7,189 [+ or -] 545      5,292 [+ or -] 282       0.007
CD62L         63,591 [+ or -] 4,614   80,656 [+ or -] 5,954     0.032

(a) Includes subjects from UPREST and UPDOSE (n= 24).
Source of some immunofluorescence markers differed for
UP50, resulting in changes in baseline values, so these
healthy subjects were not included.

Table 8. Summary of UFP exposure effects.

                                        Lymphocyte
                                        subsets and     Leukocyte
Protocol        Adhesion molecules      activation      counts

UPREST (n=12)   No convincing effects   No effects      No effects
                (see text)
UPDOSE (n=12)   Decreased monocyte      Increased       Decreased
                CD54                    CD[25.sup.+]    monocytes
                Decreased PMN T         and basophils
                cells (females)         (females)
                CD49d (males)
UP50            Decreased monocyte      Increased       Decreased
(n=16)          CD 18 and CD54          CD[25.sup.+]    eosinophils
                (males)                 T cells
                Decreased PMN CD18
                and increased CD11
                a
UPASTHMA        Decreased monocyte      Decreased       Decreased
(n=16)          CID11b                  CD[4.sup.+]     eosinophils
                Decreased PMN CD54      T cells         and basophils
                and increased CD62L
                (males)
                Decreased eosinophil
                CD11b